A seed is an embryonic plant enclosed in a protective outer covering called the seed coat. In monocots, the seed coat is fused with the pericarp, which is composed of multiple layers of cells and contains genetic code from the maternal parent only. As such, pericarp tissue can be used to identify the maternal lineage of a seed. Paternal lineage can be further derived from the pericarp genotype in combination with the seed (embryo or endosperm) genotype, which contains the genetic information from both maternal and paternal background. Determining lineage is important for germplasm security to ensure that proprietary parental lines remain proprietary.
Tissues or other adhering structures connect the pericarp to the interior part of the seed, mainly endospermic tissue. This, coupled with the relatively small size of most seeds, can make separation and removal of pericarp difficult, or at least time consuming and laborious. Molecular analysis of impure pericarp tissue contaminated with interior seed tissue (e.g. endosperm), which carries both maternal and paternal genetic information, leads to ambiguous genetic identification of the maternal lineage. Therefore, an efficient pericarp removal and purifying method is required prior to molecular analysis.
Pericarp removal processes have been developed and used in the art for both food processing and pericarp molecular analysis. Some pericarp removal methods involve soaking corn kernels in a chemical solution (e.g. sodium hydroxide (NaOH) and/or hydrogen peroxide (H2O2)) for a relatively long period of time (e.g. hours). Such chemical soaking has been found to substantially loosen pericarp such that an automated method of separation can be used to separate the loosened pericarp from the endosperm. The methods allow batches of a substantial number of kernels to be processed. However, such processes require chemicals, as well as costs and handling associated with them. Most importantly, the chemicals used combined with the significant amount of time of soaking may damage or otherwise affect the DNA in the pericarp.
To avoid damage to DNA when separating pericarp from the remainder of the kernel for molecular analysis, current conventional methodologies have stayed away from chemical steeping. Instead, current methods involve trying to loosen the pericarp by soaking the kernels overnight in distilled water instead of chemicals. After such soaking, the pericarp is manually cut or picked off. Water soaking is not as effective as chemicals at loosening the pericarp.
To accurately identify parental lineage, a high yield of DNA is required for molecular analysis such as genome-wide genetic analysis. To obtain sufficient DNA for one sample using prior art methods, multiple seeds (usually on the order of 10 to 100 or so) have been needed. Manual removal of the pericarp from a sufficient number of individual seeds can take one, two, or more person-hours. Furthermore, it has been difficult, if not impossible, to completely remove all interior seed tissue, such as endospermic tissue, from the pericarp with the water soaking method, which makes it difficult to achieve the purity level needed or desired for accurate molecular analysis. In addition, the pooled DNA from multiple seeds, particularly from commercial seeds, brings a potential risk of contamination due to the mixing of seed source. To avoid such contamination and reduce the labor force, using a single seed for one sample would be desirable for molecular analysis.
There is a need for an improvement in the art of pericarp removal and tissue purification for accurate molecular analysis of pericarp tissue. Moreover, there is a need for the pericarp tissue sample to comprise tissue from fewer seeds, or even a single seed.